A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction.

Naresh Polisetti,Philip Maier,Günther Schlunck,Benjamin Roschinski,Ursula Schlötzer-Schrehardt,Thomas Reinhard

doi:10.3390/ijms221810067

Abstract

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.

Highlights

Homeostasis of the corneal epithelium, which is an important prerequisite for corneal transparency and visual function, is maintained by the reservoir of limbal epithelial stem/progenitor cells (LEPCs) located in the basal epithelial layer of the corneoscleral limbus [1]
The efficiency of limbal tissue decellularization was initially screened by hematoxylin and eosin (H&E) staining to assess cellularity
Sections of decellularized human limbus (DHL) in the presence or absence of dextran were cell-free and the collagen fibrils of the extracellular matrix (ECM) were still arranged in a regular pattern (Figure 1A)

Summary

Introduction

Homeostasis of the corneal epithelium, which is an important prerequisite for corneal transparency and visual function, is maintained by the reservoir of limbal epithelial stem/progenitor cells (LEPCs) located in the basal epithelial layer of the corneoscleral limbus [1]. The transplantation of ex vivo expanded LEPCs or oral mucosal epithelial cells on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface [9,10]. It is applied in various clinical centers with a reported overall success rate of 60 to 70% [11,12,13], but long-term results beyond 10 years of follow-up are not well documented. Current research focuses on identifying limbal niche components and developing appropriate biomimetic scaffolds with low immunogenic potential to replicate the biological niche in vitro and to avoid immune responses [16,17]

Methods

Results

Conclusion